Project summary The goal of this application is to address critical gaps in our understanding of the central DNA repair pathway in humans by using new single molecule methods. In replicating mammalian cells chromosomal breaks are repaired via two main pathways: the non-homologous end-joining (NHEJ) pathway, which is active throughout the cell cycle, and homologous recombination (HR) that is mainly active during replication. Mutations in HR proteins are associated with genome instability and predisposition to cancer, whereas mutations in NHEJ proteins result in genome instability, severe combined immunodeficiency (SCID), and IR sensitivity, the latter relevant to patient hypersensitivity to therapeutic IR. Consequently, NHEJ and HR factors are recognized as promising targets for inhibition to improve the efficacy of radiation and chemotherapy. Additionally, the interplay between these pathways has implication for the development of resistance to therapy in the treatment of cancer. Although the mechanisms that control NHEJ and HR also have key roles in normal human development, our current state of knowledge regarding the organization and crosstalk between these pathways and their correlations with the DNA damage response (DDR) is minimal. We especially know very little about the physical nature of the repair complexes and how they are assembled/disassembled and regulated at DNA break sites; this is because common biochemical, structural and cell biology approaches are limited in their capacities to provide this information. In this study we will use an array of innovative single-molecule techniques and assays to address this knowledge gap and to define the molecular mechanisms of DSB repair via NHEJ and at single-ended breaks in replicating cells. We use single-molecule biochemical methods to determine the role of NHEJ filament proteins, and reexamine basic mechanisms that link DDR and NHEJ repair. We will use single-molecule localization microscopy and live cell imaging to map the organization of HR/NHEJ repair intermediates in-vivo and determine their positional dependence, and study their regulation and the role of DDR factors in pathway choice. We will define the nanoscale architecture of DSB complexes in cells and determine their association with cellular DNA damage response (DDR) factors and characterize how these are modulated in different types of DSBs. Combined, the proposed study will address critical unanswered questions with have enormous potential for advancing the field of DNA damage research.